%0 Journal Article %J The Journal of Steroid Biochemistry and Molecular Biology %D 2007 %T Computational analysis of the active sites in binary and ternary complexes of the vitamin D receptor %A Wanda Sicinska %A Piotr Rotkiewicz %K Binding Sites %K Biomimetic Materials %K Computer Simulation %K Models, Molecular %K Peptides %K Protein Binding %K Protein Structure, Tertiary %K Receptors, Calcitriol %K Solvents %X We have developed a program CCOMP that compares overlapping fragments of two protein complexes and identifies differently oriented amino acids. CCOMP initially performs a sequence alignment of the analyzed receptors, then superimposes the corresponding aligned residues, and finally calculates the root mean square deviation (RMSD) of individual atoms, every amino acid and the entire complex. Thus, amino acids important for functional differences between both complexes can be detected. Application of CCOMP to 1alpha,25-(OH)(2)D(3)-hVDR (1DB1) [Proc. Natl. Acad. Sci. U.S.A. 98 (2001) 5491] and 1alpha,25-(OH)(2)D(3)-rVDR-peptide (1RK3) [Biochemistry 43 (2004) 4101] complexes revealed that the peptide (KNHPMLMNLLKDN) mimicking a co-activator sequence significantly changes the side chain conformation of 35 amino acids. Four of these residues (K242, I256, K260, E416) actually contact the peptide, but all of them are essential for biological activity. Only two (L309 and L400) of the 35 differently oriented amino acids contact the ligand. Interestingly, when the peptide is present (1RK3) leucine 400 shifts closer (0.7A) to the vitamin D 26-methyl group. Applying the CCOMP and DSSP programs to binary and ternary VDR complexes also resulted in establishing that seven amino acids (I238, S252, I256, L413, L415, E416, V417) exhibit significant differences in solvent accessibility and are capable of interacting with co-activators. %B The Journal of Steroid Biochemistry and Molecular Biology %V 103 %P 305-9 %8 2007 Mar %G eng %N 3-5 %R 10.1016/j.jsbmb.2006.12.077 %0 Journal Article %J Cancer Research %D 2005 %T DNA vaccine expressing the mimotope of GD2 ganglioside induces protective GD2 cross-reactive antibody responses %A Elizabeth Bolesta %A Aleksandra Kowalczyk %A Andrzej Wierzbicki %A Piotr Rotkiewicz %A Barbara Bambach %A Chun-Yen Tsao %A Irena Horwacik %A Andrzej Koliński %A Hanna Rokita %A Martin Brecher %A Xinhui Wang %A Soldano Ferrone %A Danuta Kozbor %K Active %K Active: methods %K Amino Acid Sequence %K Animals %K Antibodies %K Antibody %K Binding Sites %K Cancer Vaccines %K Cancer Vaccines: genetics %K Cancer Vaccines: immunology %K Cancer Vaccines: pharmacology %K Cross Reactions %K DNA %K DNA: genetics %K DNA: immunology %K DNA: pharmacology %K Female %K Gangliosides %K Gangliosides: genetics %K Gangliosides: immunology %K Humans %K Immunoglobulin G %K Immunoglobulin G: biosynthesis %K Immunoglobulin G: immunology %K Immunotherapy %K Inbred BALB C %K Melanoma %K Melanoma: immunology %K Melanoma: therapy %K Mice %K Molecular Sequence Data %K Monoclonal %K Monoclonal: genetics %K Monoclonal: immunology %K Neuroblastoma %K Neuroblastoma: immunology %K Neuroblastoma: therapy %K Peptide Library %K Peptides %K Peptides: genetics %K Peptides: immunology %K SCID %K Vaccines %K Xenograft Model Antitumor Assays %X The GD2 ganglioside expressed on neuroectodermally derived tumors, including neuroblastoma and melanoma, is weakly immunogenic in tumor-bearing patients and induces predominantly immunoglobulin (Ig)-M antibody responses in the immunized host. Here, we investigated whether interconversion of GD2 into a peptide mimetic form would induce GD2 cross-reactive IgG antibody responses in mice. Screening of the X(15) phage display peptide library with the anti-GD2 monoclonal antibody (mAb) 14G2a led to isolation of mimetic peptide 47, which inhibited the binding of 14G2a antibody to GD2-positive tumor cells. The peptide was also recognized by GD2-specific serum antibodies from a patient with neuroblastoma, suggesting that it bears an internal image of GD2 ganglioside expressed on the tumor cells. The molecular basis for antigenicity of the GD2 mimetic peptide, established by molecular modeling and mutagenesis studies, led to the generation of a 47-LDA mutant with an increased mimicry to GD2. Immunization of mice with peptide 47-LDA-encoded plasmid DNA elicited GD2 cross-reactive IgG antibody responses, which were increased on subsequent boost with GD2 ganglioside. The vaccine-induced antibodies recognized GD2-positive tumor cells, mediated complement-dependent cytotoxicity, and exhibited protection against s.c. human GD2-positive melanoma growth in the severe combined immunodeficient mouse xenograft model. The results from our studies provide insights into approaches for boosting GD2 cross-reactive IgG antibody responses by minigene vaccination with a protective epitope of GD2 ganglioside. %B Cancer Research %V 65 %P 3410–8 %8 apr %@ 7168458906 %G eng %U http://www.ncbi.nlm.nih.gov/pubmed/15833876 %R 10.1158/0008-5472.CAN-04-2164 %0 Journal Article %J Journal of Computer-Aided Molecular Design %D 2003 %T Protein fragment reconstruction using various modeling techniques %A Michal Boniecki %A Piotr Rotkiewicz %A Jeffrey Skolnick %A Andrzej Koliński %K Amino Acid Sequence %K Binding Sites %K Hydrogen Bonding %K Models %K Molecular %K Peptide Fragments %K Peptide Fragments: chemistry %K Protein Conformation %K Protein Structure %K Proteins %K Proteins: chemistry %K Secondary %X Recently developed reduced models of proteins with knowledge-based force fields have been applied to a specific case of comparative modeling. From twenty high resolution protein structures of various structural classes, significant fragments of their chains have been removed and treated as unknown. The remaining portions of the structures were treated as fixed - i.e., as templates with an exact alignment. Then, the missed fragments were reconstructed using several modeling tools. These included three reduced types of protein models: the lattice SICHO (Side Chain Only) model, the lattice CABS (Calpha + Cbeta + Side group) model and an off-lattice model similar to the CABS model and called REFINER. The obtained reduced models were compared with more standard comparative modeling tools such as MODELLER and the SWISS-MODEL server. The reduced model results are qualitatively better for the higher resolution lattice models, clearly suggesting that these are now mature, competitive and complementary (in the range of sparse alignments) to the classical tools of comparative modeling. Comparison between the various reduced models strongly suggests that the essential ingredient for the sucessful and accurate modeling of protein structures is not the representation of conformational space (lattice, off-lattice, all-atom) but, rather, the specificity of the force fields used and, perhaps, the sampling techniques employed. These conclusions are encouraging for the future application of the fast reduced models in comparative modeling on a genomic scale. %B Journal of Computer-Aided Molecular Design %V 17 %P 725–38 %8 nov %G eng %U http://www.ncbi.nlm.nih.gov/pubmed/15072433 %0 Journal Article %J Journal of Medicinal Chemistry %D 2002 %T 2-Ethyl and 2-ethylidene analogues of 1alpha,25-dihydroxy-19-norvitamin D(3): synthesis, conformational analysis, biological activities, and docking to the modeled rVDR ligand binding domain. %A Rafal R. Sicinski %A Piotr Rotkiewicz %A Andrzej Koliński %A Wanda Sicinska %A Jean M. Prahl %A Connie M. Smith %A Hector F. DeLuca %K Animals %K Binding Sites %K Biological Transport %K Calcitriol %K Calcium %K Cell Differentiation %K Chromatography, High Pressure Liquid %K HL-60 Cells %K Humans %K Intestinal Mucosa %K Ligands %K Magnetic Resonance Spectroscopy %K Male %K Models, Molecular %K Molecular Conformation %K Rats %K Receptors, Calcitriol %K Spectrophotometry, Ultraviolet %K Structure-Activity Relationship %K Swine %X Novel 19-nor analogues of 1alpha,25-dihydroxyvitamin D(3) were prepared and substituted at C-2 with an ethylidene group. The synthetic pathway was via Wittig-Horner coupling of the corresponding A-ring phosphine oxides with the protected 25-hydroxy Grundmann's ketones. Selective catalytic hydrogenation of 2-ethylidene analogues provided the 2alpha- and 2beta-ethyl compounds. The 2-ethylidene-19-nor compounds with a methyl group from the ethylidene moiety in a trans relationship to the C(6)-C(7) bond (E-isomers) were more potent than the corresponding Z-isomers and the natural hormone in binding to the vitamin D receptor. Both geometrical isomers (E and Z) of (20S)-2-ethylidene-19-norvitamin D(3) and both 2alpha-ethyl-19-norvitamins (in the 20R- and 20S-series) have much higher HL-60 differentiation activity than does 1alpha,25-(OH)(2)D(3). Both E-isomers (20R and 20S) of 2-ethylidene vitamins are characterized by very high calcemic activity in rats. The three-dimensional structure model of the rat vitamin D receptor and the computational docking of four synthesized (20R)-19-norvitamin D(3) analogues into its binding pocket are also reported. %B Journal of Medicinal Chemistry %V 45 %P 3366-80 %8 2002 Aug 1 %G eng %N 16 %0 Journal Article %J Proteins %D 2001 %T Model of three-dimensional structure of vitamin D receptor and its binding mechanism with 1alpha,25-dihydroxyvitamin D(3) %A Piotr Rotkiewicz %A Wanda Sicinska %A Andrzej Koliński %A Hector F. DeLuca %K Amino Acid %K Amino Acid Sequence %K Animals %K Binding Sites %K Calcitriol %K Calcitriol: chemistry %K Calcitriol: genetics %K Computational Biology %K Humans %K Ligands %K Models %K Molecular %K Molecular Sequence Data %K Point Mutation %K Protein Conformation %K Protein Structure %K Rats %K Receptors %K Sequence Homology %K Tertiary %X Comparative modeling of the vitamin D receptor three-dimensional structure and computational docking of 1alpha,25-dihydroxyvitamin D(3) into the putative binding pocket of the two deletion mutant receptors: (207-423) and (120-422, Delta [164-207]) are reported and evaluated in the context of extensive mutagenic analysis and crystal structure of holo hVDR deletion protein published recently. The obtained molecular model agrees well with the experimentally determined structure. Six different conformers of 1alpha,25-dihydroxyvitamin D(3) were used to study flexible docking to the receptor. On the basis of values of conformational energy of various complexes and their consistency with functional activity, it appears that 1alpha,25-dihydroxyvitamin D(3) binds the receptor in its 6-s-trans form. The two lowest energy complexes obtained from docking the hormone into the deletion protein (207-423) differ in conformation of ring A and orientation of the ligand molecule in the VDR pocket. 1alpha,25-Dihydroxyvitamin D(3) possessing the A-ring conformation with axially oriented 1alpha-hydroxy group binds receptor with its 25-hydroxy substituent oriented toward the center of the receptor cavity, whereas ligand possessing equatorial conformation of 1alpha-hydroxy enters the pocket with A ring directed inward. The latter conformation and orientation of the ligand is consistent with the crystal structure of hVDR deletion mutant (118-425, Delta [165-215]). The lattice model of rVDR (120-422, Delta [164-207]) shows excellent agreement with the crystal structure of the hVDR mutant. The complex obtained from docking the hormone into the receptor has lower energy than complexes for which homology modeling was used. Thus, a simple model of vitamin D receptor with the first two helices deleted can be potentially useful for designing a general structure of ligand, whereas the advanced lattice model is suitable for examining binding sites in the pocket. %B Proteins %V 44 %P 188–199 %8 2001 %G eng %U http://www.ncbi.nlm.nih.gov/pubmed/11455592 %0 Journal Article %J Protein Engineering %D 2001 %T Three-dimensional modeling of the I-TevI homing endonuclease catalytic domain, a GIY-YIG superfamily member, using NMR restraints and Monte Carlo dynamics %A Janusz M. Bujnicki %A Piotr Rotkiewicz %A Andrzej Koliński %A Leszek Rychlewski %K Algorithms %K Binding Sites %K Biomolecular %K Endodeoxyribonucleases %K Endodeoxyribonucleases: chemistry %K Models %K Molecular %K Monte Carlo Method %K Nuclear Magnetic Resonance %K Protein Structure %K Sequence Alignment %K Tertiary %X Using a recent version of the SICHO algorithm for in silico protein folding, we made a blind prediction of the tertiary structure of the N-terminal, independently folded, catalytic domain (CD) of the I-TevI homing endonuclease, a representative of the GIY-YIG superfamily of homing endonucleases. The secondary structure of the I-TevI CD has been determined using NMR spectroscopy, but computational sequence analysis failed to detect any protein of known tertiary structure related to the GIY-YIG nucleases (Kowalski et al., Nucleic Acids Res., 1999, 27, 2115-2125). To provide further insight into the structure-function relationships of all GIY-YIG superfamily members, including the recently described subfamily of type II restriction enzymes (Bujnicki et al., Trends Biochem. Sci., 2000, 26, 9-11), we incorporated the experimentally determined and predicted secondary and tertiary restraints in a reduced (side chain only) protein model, which was minimized by Monte Carlo dynamics and simulated annealing. The subsequently elaborated full atomic model of the I-TevI CD allows the available experimental data to be put into a structural context and suggests that the GIY-YIG domain may dimerize in order to bring together the conserved residues of the active site. %B Protein Engineering %V 14 %P 717–721 %8 oct %G eng %U http://www.ncbi.nlm.nih.gov/pubmed/11739889